Secretion of heat shock ‐60, ‐70 kD protein, IL‐1β and TNFα levels in serum of a term normal pregnancy and patients with pre‐eclampsia development

Abstract The extracellular heat shock proteins (eHsp) family act as molecular chaperones regulating folding, transporting protein and are associated with immune modulation in different physiological and pathological processes. They have been localized in different gestational tissues and their concentration in amniotic fluid and serum has been determined. In the present study, we proposed to determine the concentration of eHsp‐60, ‐70, IL‐1β and TNFα in the serum of pregnant patients with 34 weeks of gestation with and without clinical evidences of preeclampsia (PE). Our results indicate significant increase of these markers in patients with PE with respect to healthy pregnant patients without active labor. Finally, the concentration of eHsp‐60 and ‐70 correlated positively with the hepatic dysfunction markers uric acid, lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT) glutamic pyruvic transaminase (GPT), and inflammatory IL‐1β and TNFα response. In conclusion, our results demonstrate a strong associated between Hsp and marker of hepatic dysfunction.

remodelling of maternal spiral arteries results in a deficient maternal blood supply to the placenta, endothelial dysfunction, and systemic inflammatory cytokines. 3 Within this endothelial dysfunction environment provoked by an inadequate syncytiotrophoblast remodelling have been detected extracellular heat-shock proteins (eHsp).
The eHsp are highly conserved molecules that regulate cellular homoeostasis, proliferation and differentiation of the immunological cell. 4 The eHsp can be released to extracellular space in response to cellular stress by non-classical protein transport mechanisms. Molvarec et al 5

| Patients
This study was reviewed and approved by the National Institute of Perinatology Ethics and Research Committees (registration number 212250-3210091). All patients were explained the purpose of the study, and informed consent was obtained. This study included 140 pregnant patients with no obstetric complications and no prior history of PE. Controls group (n = 78) was divided into two categories: patients with 34-weeks of pregnancy (n = 28; the same gestational age as the group of PE) and term in labour (n = 50; with gestational age to term ≥37) defined as dilation of cervical canal (≥4 cm) and uterine contraction sustained. Patients who development clinical data of severe PE that came to the emergency unit (n = 62) was defined according to the American College of Obstetricians and Gynecologists guidelines. 8

PE patients
were included in the study previous of the therapeutic treatment.
In all cases, blood samples of all patients were taken in only one occasion. The serum was obtained from 5 millilitres of peripheral maternal blood samples and stored at −80°C for the quantification of the eHsp-60, -70, IL1-β and TNFα by specific immunoassay ELISA.

| Biochemical assays
Commercial ELISA kits were used to measure concentration of eHsp-

| Statistical analysis
Proportional data were analysed using one-way ANOVA, and significant difference between groups were determined by Tukey′s test.
The Spearman rank correlation (r) between eHsp and IL-1β, TNFα and clinical parameters were determined. All the assays were independently replicated at least three times, and the data were presented as mean ± SEM. Significant difference was accepted at P value ≤0.05.

| RESULTS AND DISCUSSION
Abnormal morphogenesis of the placenta is the main cause for the development of PE, which is associated with spatial and temporal secretion of various markers of endothelial damage and anti-angiogenic factors. It has been demonstrated that molecules secreted into the cell as "alarm system" are eHsp which modulate different cellular process and pathological conditions. 10 In this study, we found an increase significantly in the term labour group with respect to 34-weeks′pregnancy by 1.  (Table 1) Figure 1E).
Interestingly, induce also expression of protein inhibitor α, a negative regulator of the classical nuclear transcription factor-kappa B (NFκβ) pathway. 10 In this study, we found that in patients with 34-week pregnant, the secretion profile of Hsp-70 was 1.6 ± 0.12 ng/mL (Figure 1

CONFLI CT OF INTEREST
The authors declare that they have no competing interests to disclose.

AUTHOR CONTRI BUTIONS
MCAC, ABG and MOC identified patients with clinical evidence of PE and obtained the blood samples from the three study groups.
MCAC, EBG, GGL carried out the determination of heat shock protein and inflammatory cytokine. JFA perform the correlation analysis.
MCAC, OFH, NFD, AMH and HFH were involved with the study design. HFH obtained the funding and writing the manuscript. NFD and JFA discussed the manuscript in its final form. All authors read and approval the final manuscript.